氧氟沙星与脲诱导牛血清白蛋白结合的机制研究

摘要 利用荧光光谱和紫外光谱研究了脲（Urea）对牛血清白蛋白（BSA）结构的影响以及氧氟沙星（Oflxacin）与脲诱导的BSA结合的情况。结果显示：Urea诱导BSA变性历经两步、三态过程,且伴随中间态的形成。随着Urea浓度的增大,BSA荧光强度降低并先蓝移（344 nm～336 nm）,后又红移至350 nm。Urea浓度在4.6～5.2 mol/L范围时,Oflx对BSA中间态有强的猝灭作用（KQ=10.46×104 L/mol, Urea 4.8 mol/L）和较大的结合常数（KA=3.8807×105 L/mol, Urea 4.8 mol/L）,但是结合位点数小（n=0.76, Urea 5.0 mol/L）,能量传递效率低（E=0.3002, Urea 4.8 mol/L）。同步荧光光谱显示：Urea诱导BSA去折叠时,Trp-212残基微环境并未发生改变,而Tyr的最大荧光发射峰蓝移,Oflx的加入诱导Trp-212的微环境更具疏水性。Oflx加速了Urea对BSA的失活作用。     

荧光相图法研究脲诱导牛血清白蛋白的去折叠过程

作为从分子水平上阐明生命奥秘的中心课题之一,蛋白质的折叠问题一直受到生物化学、生物物理学和结构生物学等领域研究工作者的高度关注。在蛋白质的变性过程中,它们往往达不到完全去折叠,而是会形成不同的部分折叠中间态[1-3],这些部分折叠中间态在蛋白质折叠过程中起着重要作     

脲和盐酸胍诱导溶菌酶去折叠的荧光相图法研究

用荧光相图法分别研究了脲和盐酸胍诱导卵清溶菌酶去抓叠的过程。当变性体 系中无还原剂2－巯基乙醇存在、脲浓度从0变化至4.0 mol/L（或盐酸胍浓度从0变 化至3.0 mol/L）时,溶菌酶从天然态转变为部分折叠中间态,当脲浓度从4.0 mol/L变化至8.0 mol/L（或盐酸胍浓度从3.0 mol/L变化至6.0 mol/L）时,溶菌 酶从中间态转变为去折叠态,此时该蛋白的变性过程符合“三态模型”。而当变性 体系中有该还原剂存在时,溶菌酶则由天然态直接转变为去折叠态,此时脲诱导该 蛋白去折叠的过程符合曲型的“二态模型”。实难结果表明荧光相图法可以检测蛋 白南去抓叠的中间态。     

脲和盐酸胍诱导过氧化氢酶去折叠的研究

用荧光相图法分别研究了脲和盐酸胍诱导牛肝过氧化氢酶去折叠的过程。当脲 浓度从0依次增大至0.50,4.5和8.0 mol/L时,过氧化氢酶从天然四聚体依次转变 为蓬松的四聚体、部分折叠的无活性二聚体和去折叠态,而当盐酸胍浓度从0依次 变化至0.65,2.5和6.0 mol/L时,过氧化氢酶则从天然四聚体集资转变为部分折叠 的激活二聚体、部分折叠的单体和去折叠态,这表明无论是用脲还是用盐酸胍作为 变性剂,该蛋白的变性过程都符合“四态模型”,但这两种变性剂诱导该蛋白去折 叠的途径和机制有较大差异。实验结果表明荧光相图法可以检测蛋白质去折叠的中 间态。用等温滴定量去热法研究了盐酸胍诱导过氧化氢酶去折叠过程的热力学, 25.0 ℃时低浓度盐酸胍诱导该蛋白从天然四聚体转变为部分折叠的激活二聚体的 本征摩尔构象变化焓、Gibbs自由能和熵分别为-69.2 kJ·mol~(-1),6.43 kJ· mol~(-1)和-254 J·K~(-1)·mol~(-1),据此推断盐酸胍通过熵效应和静电效应来 稳定和激活该二聚体。     

4-(1H-imidazol-1-yl) aniline: A new ligand of mixed-mode chromatography for antibody purification

4-(1H-imidazol-1-yl) aniline (AN) was immobilized on Sepharose CL-6B (AN-Sepharose) for use as a new ligand of mixed-mode chromatography. Adsorption equilibria of immunoglobulin G (IgG) and bovine serum albumin (BSA) to AN-Sepharose were studied at extensive pH values (4.0–8.8) and salt concentrations (0–1.0 mol/L). Static binding studies indicated that AN-Sepharose had a good salt-tolerance property for IgG adsorption up to 1.0 mol/L NaCl. This was attributed to the combined ligand–protein interactions (hydrophobic interaction, hydrogen bonding and charge transfer interaction). By contrast with BSA, AN-Sepharose showed a high binding selectivity for IgG at NaCl > 0.2 mol/L. Dynamic binding capacities (DBC) of IgG and BSA at 10% breakthrough were measured at pH 4.0–8.8 by frontal analysis chromatography. IgG had DBC values over 40 mg/mL at pH 7.0–8.8, and the maximum reached 59 mg/mL at pH 8.0. At pH 5.0, a distinct drop in DBC to 8.5 mg/mL was observed, but that for BSA kept over 22 mg/mL. The result suggested that IgG could be selectively desorbed from AN-Sepharose by decreasing pH to about 5. Therefore, compared to BSA, AN-Sepharose exhibited a dual-selectivity for IgG in both adsorption and elution. Purification of IgG from bovine serum also confirmed the dual-selectivity. IgG purity of the pooled fractions by elution at pH 4.0, 4.5 and 5.0 reached 55% and the highest purity, 80%, was obtained at pH 4.5. The average purification factor of IgG was over 25. The results indicate that AN is a promising ligand of mixed-mode chromatography for antibody purification from a complex feedstock.     

Hydrophobic Interaction Between Domain I of Albumin and B Chain of Detemir May Support Myristate-Dependent Detemir-Albumin Binding

The bindings of detemir [LysB29(Nε-tetradecanoyl)des(B30)-insulin] with two highly homologous albumins, HSA (human serum albumin) and BSA (bovine serum albumin), were investigated through CD, spectrofluorophotometry, and molecular docking analysis. The absence of any tryptophanyl residue in detemir makes albumin binding study possible by exclusive tryptophanyl spectral quenching at 340 nm (λem = 296 nm). The interactions found to be static (Kq > 10¹⁰ M^?1 s^?1) with Stern–Volmer constants ≈10³ M^?1. The observed ΔG ⁰ that was negative in all cases concludes the reactions were spontaneous. Domains I and III of an albumin unfold with 5.0 M urea at pH 7.4, although domain II remains intact. Significant decreases in ΔH ⁰ and ΔS ⁰ were due to unfolding explicit that detemir binding may involve domains I and III of albumins. Temperature-dependent changes in binding were higher in HSA than BSA but after unfolding such changes were very less, further indicating the role of domains I and III in detemir binding. Pro28 and Tyr26 of insulin were found to be interacting with Arg114 and Val116 of HSA domain I, while myristate segment of detemir binds to Lys519 of domain III. Interactions seem to be predominantly hydrophobic and entropy driven. Although detemir binds to albumin through myristate, the peptide part shows involvement in binding.     

The effects of urea and n-propanol on collagen denaturation: using DSC, circular dicroism and viscosity

The effect of urea and n-propanol on circular dichroism (CD) and viscosity of purified type1 collagen solution at various temperatures and differential scanning calorimetry (DSC) of rat-tail tendon (RTT) collagen fibre have been studied. CD reveals a spectrum with a positive peak at around 220 nm and a negative peak at 200 nm characteristics of collagen triple helix. The molar ellipticity decreases as the concentration of urea increases up to particular concentration (collagen solution treated with 265 μM of urea) and after that it increases (collagen solution treated with 500 μM of urea). There is a linear decrease in molar ellipticity as the concentration of n-propanol increases. Denaturation temperature of urea and n-propanol treated with purified collagen solution has been studied using viscosity method. Additives such as urea and n-propanol decrease the thermal stability of collagen triple helix in solution and in RTT collagen fibre. Thermal helix to coil transition of urea and n-propanol treated collagen depends on the degree of hydration and the concentration of these additives. Thermodynamic parameters such as the peak temperature, enthalpy of activation, and energy of activation for collagen-gelatin transition for native, urea and n-propanol treated RTT collagen fibre has been calculated using DSC. The change in the thermodynamic parameters has been observed for native, urea and n-propanol treated RTT collagen fibres. The experimental results show that the change in the water structure, dehydration and desolvation induced by different additives such as urea and n-propanol on RTT may vary with the type of denaturation.     

荧光光谱法研究牛血清蛋白在盐酸胍体系中的变性

应用荧光光谱技术,对盐酸胍与牛血清蛋白在30℃水溶液中的结合作用及造成牛血清蛋白变性的过程进行了研究,考察了盐酸胍诱导牛血清蛋白变性时荧光强度和峰位的变化规律,并计算出伸展分数fu,变性平衡常数Ku,伸展吉布斯自由能△Gu,衡量蛋白质对变性剂稳定性的参量△GH2o,衡量蛋白质变性协同性的参量m和变性中点C1/2.研究结...     

Hydrophobic interaction chromatography coupled with atomic fluorescence spectrometric detection Effect of the denaturation on the determination of thiolic proteins

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol dm⁻³ urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0 M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO₃ in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg²⁺, present in solution as Hg²⁺-urea complex, is selectively detected by AFS in a Ar/H₂ miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100±2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and β-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10⁻¹⁰-10⁻¹² mol dm⁻³, depending on the considered protein, with linear calibration curves spanning over four decades of concentration.     

Investigation of the association behaviors between biliverdin and bovine serum albumin by fluorescence spectroscopy

The interaction between biliverdin and bovine serum albumin (BSA) has been studied by steady fluorescence spectroscopy, synchronous fluorescence and resonance light scanning spectra. The binding of biliverdin to BSA quenches the tryptophan residue fluorescence and the results show that both static and dynamic quenching occur together with complex formation. The binding constant and binding sites of biliverdin to BSA at pH 7.1 are calculated to be 3.33 × 10⁸ L/mol and 1.54, respectively, according to the double logarithm regression curve. In addition, the distance between the biliverdin and BSA is estimated to be 1.25 nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues has not obvious changes, which obeys the phase distribution model. Finally, the thermodynamic data show that biliverdin molecules enter the hydrophobic cavity of BSA via hydrophobic interaction.     

Thermal Stability of Immobilised α-chymotrypsinogen

The unfolding of α-chymotrypsinogen covalently immobilized on silica beads has been studied by differential scanning calorimetry (DSC). The enzyme undergoes an unfolding transition which, unlike the free protein, cannot be approximated by a single two-state process. After immobilization, the unfolding is characterized by the presence of two partially overlapping transitions, both of them show two-state behavior. The two processes correspond to the separate unfolding of the two domains of the α-chymotrypsinogen molecule. The loss of cooperativity behavior is a consequence of the covalent immobilization. The two domains showed different thermal stability as functions of pH. One of them unfolded with a transition temperature T _m2 higher than T _m of the free enzyme, implying stabilization effect of immobilization. However, below pH 4.5, its native structure is lost. The other transition shows a remarkable pH-independent thermal stability from pH 2.5 to 7.0. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparation of gold nanoparticles on eggshell membrane and their biosensing application

A facile green biosynthesis method has been successfully developed to prepare gold nanoparticles (AuNPs) of various core sizes (25 ± 7 nm) using a natural biomaterial, eggshell membrane (ESM) at ambient conditions. In situ synthesis of AuNPs-immobilized ESM is conducted in a simple manner by immersing ESM in a pH 6.0 aqueous solution of HAuCl₄ without adding any reductant. The formation of AuNPs on ESM protein fibers is attributed to the reduction of Au(III) ions to Au(0) by the aldehyde moieties of the natural ESM fibers. Energy dispersive X-ray spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray powder diffraction unambiguously identify the presence of AuNPs on ESM. The effect of pH on the in situ synthesis of AuNPs on ESM has been investigated in detail. The pH of the gold precursor (HAuCl₄) solution can influence the formation rate, dispersion and size of AuNPs on ESM. At pH ≤3.0 and ≥7.0, no AuNPs are observed on ESM while small AuNPs are homogeneously dispersed on ESM at pH 4.0-6.0. The optimal pH for AuNPs formation on ESM is 6.0. AuNPs/ESMs are used to immobilize glucose oxidase (GO_x) for glucose biosensing. AuNPs on ESM can increase the enzyme activity of GO_x. The linear response range of the glucose biosensor is 20 μM to 0.80 mM glucose with a detection limit of 17 μM (S/N = 3). The biosensor has been successfully applied to determine the glucose content in commercial glucose injections. Our work provides a very simple, non-toxic, convenient, and green route to synthesize AuNPs on ESM which is potentially useful in the biosensing field.     

Thiol-functionalized shell crosslinked knedel-like (SCK) nanoparticles: a versatile entry for their conjugation with biomacromolecules

Shell crosslinked knedel-like (SCK) nanoparticles were prepared having thiol-terminated poly(ethylene glycol) (PEG) chains extending throughout their shell layers and were then conjugated with bovine serum albumin (BSA) as a model biomacromolecule. The SCKs originated from amphiphilic block copolymers of acrylic acid and styrene, PAA₆₆-b-PS₇₁, pre-functionalized with ca. five mono-tert-Boc-protected diamino PEG₃₂ per polymer chain, which then had undergone deprotection and amidation with N-succinimidyl-S-acetylthiohexanionate to introduce an acetyl-protected thiol chain terminus on the end of each PEG graft. Assembly of these amphiphilic graft block copolymers into micelles, by transitioning from N,N-dimethylformamide to water, was followed by amidation-based crosslinking throughout the shell layer, with the introduction of 2,2′-(ethylenedioxy)-bis(ethylamine) and 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide methiodide, to afford SCKs bearing the acetyl-protected thiol groups. Deprotection in aqueous buffer solution by reaction with hydroxylamine hydrochloride gave the SCKs presenting a nominal number of ca. 750 thiols per nanoparticle. The solution was assayed by Ellman's method resulting in a concentration of 55±6 μM [HS], theoretical of concentration 58 μM [HS], after which the coupling with BSA was performed immediately. Tetramethylrhodamine-labeled, maleimido-functionalized BSA was allowed to react with the thiol-functionalized SCKs at stoichiometries of ca. 10, 20, and 30 BSAs/SCK, after which UV-vis spectroscopy and Bradford's assay determined a coupling efficiency of >50-60%. The SCK particle diameters were measured by TEM to be 16 nm and 20 nm and their hydrodynamic diameters were measured by dynamic light scattering to be 20 nm and 30 nm, before and after BSA conjugation, respectively.     

Protein determination using methylene blue in a synchronous fluorescence technique

A new method for detecting protein by synchronous fluorescence enhancement was developed, based on the combination of near infrared (NIR) fluorescence and the dedimerization phenomenon of methylene blue (MB). Under analytical conditions, there are linear relationships between the enhancing extent of synchronous fluorescence of MB-sodium dodecyl benzene sulfonate (SDBS)-protein at 667 nm and the concentration of protein in the range of 8.0 × 10⁻⁸-4.0 × 10⁻⁵ g mL⁻¹ for bovine serum albumin (BSA), 1.0 × 10⁻⁷-3.5 × 10⁻⁵ g mL⁻¹ for egg albumin (EA). The detection limits (S/N = 3) of BSA and EA are 8.9 ng mL⁻¹ and 10.0 ng mL⁻¹, respectively. The fluorescence enhancement mechanism is discussed in detail. Results from multiple techniques indicate that the fluorescence enhancement of the system originates from the hydrophobic microenvironment provided by BSA and SDBS, and the formation of an MB-SDBS-BSA complex, as well as the deaggregation of some MB dimer.     

Studies on the photophysical characteristics of poly(carboxylic acid)s bound protoporphyrin IX and metal complexes of protoporphyrin IX

The copolymers of methacrylic acid with protoporphyrin IX (PPIX) and the metal complexes, zinc protoporphyrin IX and magnesium protoporphyrin IX were synthesised and characterised. Corresponding acrylic acid copolymers were also synthesised. The steady state absorption and fluorescence spectral properties of the macromolecular bound fluorophores PPIX, Zn-PPIX and Mg-PPIX were investigated. Poly(methacrylic acid) bound protoporphyrin IX, zinc protoporphyrin IX and magnesium protoporphyrin IX show an increase in the fluorescence intensity and lifetime with increase in the pH in the range 2-8 with a marked transition around pH 6.0-7.0. The fluorophore concentration in the dilute solution of the copolymers is micromolar and the fluorophore to the carboxylic acid monomer ratios in the copolymer is around 10⁻³. The molecular weight of the copolymers is 100 ± 10 kD. The fluorescence decay curves of all the fluorophore bound polymers follow biexponential decay fit independent of pH. Poly(MAA-co-PPIX) and poly(MAA-co-MgPPIX) undergo well marked pH induced structural transitions in the pH range of 6.0-7.0 whereas poly(MAA-co-ZnPPIX) undergoes pH induced structural transitions in the pH range of 4.0. In the case of polyacrylic acid copolymers the changes observed in the steady state and time resolved fluorescence studies are less marked. The distinct hydrophobic and hydrophilic environments experienced by the fluorophore bound to PMMA are attributed to the dynamics of the macromolecules in dilute aqueous solutions manifested by the α-methyl group present in the copolymer. The studies carried out using the fluorophores in the time windows from 2 ns to 12 ns indicate evolving trends in the dynamic coiling and reverse coiling of poly methacrylic acid chain.     

Thermal aggregation of bovine serum albumin studied by asymmetrical flow field-flow fractionation

The use of asymmetrical flow field-flow fractionation (AsFlFFF) in the study of heat-induced aggregation of proteins is demonstrated with bovine serum albumin (BSA) as a model analyte. The hydrodynamic diameter (d_h), the molar mass of heat-induced aggregates, and the radius of gyration (R_g) were calculated in order to get more detailed understanding of the conformational changes of BSA upon heating. The hydrodynamic diameter of native BSA at ambient temperature was ∼7 nm. The particle size was relatively stable up to 60 °C; above 63 °C, however, BSA underwent aggregation (growth of hydrodynamic diameter). The hydrodynamic diameters of the aggregated particles, heated to 80 °C, ranged from 15 to 149 nm depending on the BSA concentration, duration of incubation, and the ionic strength of the solvent. Heating of BSA in the presence of sodium dodecyl sulfate (1.7 or 17 mM) did not lead to aggregation. The heat-induced aggregates were characterized in terms of their molar mass and particle size together with their respective distributions with a hyphenated technique consisting of an asymmetrical field-flow fractionation device and a multi-angle light scattering detector and a UV-detector. The carrier solution comprised 8.5 mM phosphate and 150 mM sodium chloride at pH 7.4. The weight-average molar mass (M_w) of native BSA at ambient temperature is 6.6 × 10⁴ g mol⁻¹. Incubation of solutions with BSA concentrations of 1.0 and 2.5 mg mL⁻¹ at 80 °C for 1 h resulted in aggregates with M_w 1.2 × 10⁶ and 1.9 × 10⁶ g mol⁻¹, respectively. The average radius of gyration and the average hydrodynamic radius of the heat-induced aggregate samples were calculated and compared to the values obtained from the size distributions measured by AsFlFFF. For comparison static light scattering measurements were carried out and the corresponding average molar mass distributions of solutions with BSA concentrations of 1.0 and 2.5 mg mL⁻¹ at 80 °C for 1 h gave aggregates with M_w 1.7 × 10⁶ and 3.5 × 10⁶ g mol⁻¹, respectively.     

Naked-eye colorimetric analysis of Hg2+ with bi-color CdTe quantum dots multilayer films

A novel direct quantificational method through naked-eye colorimetric analysis of Hg²⁺ was constructed based on different degree of the fluorescence quenching of bi-color quantum dots (QDs) multilayer films (2-QDMF). The functional multilayer films were assembled by layer-by-layer (LBL) deposition of oppositely charged CdTe QDs and poly(dimethyldiallylemmonium chloride) (PDDA). Then the outermost layer of 2-QDMF was cross-linked to bovine serum albumin (BSA), polyethylene glycol (PEG) or glutathione (GSH). It was found that when BSA modified quartz slides were immersed into solutions containing Hg²⁺ and Cu²⁺ respectively, the 2-QDMF can be quenched by Hg²⁺, but not by Cu²⁺. Under the optimization conditions, the quenched photoluminescence (PL) intensities of multilayer films were almost linearly proportional to the concentration of Hg²⁺ in the range of 1.0 × 10⁻⁸ to 1.0 × 10⁻⁶ mol L⁻¹ and the detection limit was 4.5 × 10⁻⁹ mol L⁻¹. The proposed method is intuitional and convenient, which can be applied to the determination of trace Hg²⁺ in the artificial water sample with satisfactory results.     

Effect of ionic strength and protein concentration on the transport of proteins through chitosan/polystyrene sulfonate multilayer membrane

The influence of ionic strength and protein concentration on the transport of bovine serum albumin (BSA), ovalbumin and lysozyme through chitosan (CHI)/polystyrenesulfonate (PSS) multilayers on polyether sulfone supports are investigated under ultrafiltration conditions. The percentage transmission and flux of BSA, ovalbumin and lysozyme were found to increase with increase in salt concentration in the protein. The percentage transmission of BSA through 9 bilayer membrane was found to increase from 5.3 to 115.6 when the salt concentration was varied from 0 to 1 M. It was observed that 0.1 M NaCl in BSA solution is capable of permeating all the BSA. When the salt concentration in BSA was further increased, a negative solute rejection (solute enrichment in permeate) was found to take place. With 9 bilayer membrane, the percentage transmission of ovalbumin was found to increase from 23.3 to 125.8 when the salt concentration in protein was increased from 0 to 0.05 M. The effect of protein concentration on protein transport is studied taking BSA as a model protein. BSA was rejected by the multilayer membrane at all the studied concentrations (0.25, 0.5, 1 and 2 mg/ml). With increase in feed concentration, maximum rejection of protein occurred at higher number of CHI/PSS bilayers. BSA solution flux was found to decrease with an increase in BSA concentration. This study indicates that it is possible to fine tune the transport properties of proteins through multilayer membranes by varying the concentration and ionic strength of protein solutions.     

Colorimetric and fluorescent chemosensor for citrate based on a rhodamine and Pb complex in aqueous solution

In this paper we unveil a novel rhodamine compound based fluorescent chemosensor (1-Pb²⁺) for colormetric and fluorescent detection of citrate in aqueous solution. This is the first fluorescent chemosensor for citrate based on rhodamine compound. The comparison of this method with some other fluorescence methods for citrate indicates that the method can detect citrate in aqueous solution by both color changes and fluorescent changes with long emission wavelength. In the new developed sensing system, 1-Pb²⁺ is fluorescent due to Pb²⁺-induced fluorescence enhancement of 1. However, the addition of citrate may release 1 into the solution with quenching of fluorescence. The chemosensor can be applied to the quantification of citrate with a linear range covering from 1.0 × 10⁻⁷ to 5.0 × 10⁻⁵ M and a detection limit of 2.5 × 10⁻⁸ M. The experiment results show that the response behavior of 1-Pb²⁺ towards citrate is pH independent in medium condition (pH 6.0–8.0). Most importantly, the fluorescence changes of the chemosensor are remarkably specific for citrate in the presence of other anions (even those that exist in high concentration), which meet the selective requirements for practical application. Moreover, the response of the chemosensor toward citrate is fast (response time less than 1 min). In addition, the chemosensor has been used for determination of citrate in urine samples with satisfactory results.     

Molecularly bonded chitosan prepared as chiral stationary phases in open-tubular capillary electrochromatography: comparison with chitosan nanoparticles bonded to the polyacrylamide phase

The chiral selector, chitosan (CS), was attached to the silanized capillary via a silane coupling agent, (3-glycidyloxypropyl)trimethoxysilane (GTS), to form the GTS-CS capillary, and results for this capillary were compared with those of a previous study on the copolymerization of CS with methacrylamide (MAA) (forming the MAA-CS capillary). The GTS-CS capillary did not exhibit enantioselectivity for d/l-tryptophan, whereas the GTS-BSA capillary, which was prepared by replacement of CS with bovine serum albumin (BSA), succeeded in the chiral separation with an Rs = 2.4 in Tris buffer (50 mM, pH 8.5). To increase CS attachment, the CS units were crosslinked by succinic acid, and the resulting GTS-CS-s capillary phase improved the resolution to 1.9. Alternatively, the SiH-CS-s capillary was constructed by CS attachment on the silicon hydride phase via stepwise silanization and hydrosilation reactions and crosslinking by succinic acid, but this approach could only achieve a resolution of 1.4 in Tris buffer (50 mM, pH 9.5). Although the GTS-CS-s and SiH-CS-s capillaries were still inferior to the MAA-CS capillary (Rs = 3.8), the enantioselectivities of the three capillaries were all in the range of 1.4-1.6. For the (±)-catechin sample, the plate heights of the GTS-CS-s and SiH-CS-s capillaries conditioned in pH 8.5 Tris buffer with 60% MeOH modifier were 0.9 cm ((−)-catechin) and 6.0 cm ((+)-catechin)) and 2.9 cm (−) and 3.2 cm (+), respectively, and these heights were comparable to the MAA-CS capillary (2.5 cm (−), 6.0 cm (+)) in pH 6.6 phosphate buffer with 80% MeOH. Finally, a racemate of ibuprofen, a weakly acidic anti-inflammatory drug, was successfully baseline resolved by the GTS-CS-s and SiH-CS-s capillaries in the borate buffers, which were 30 mM at pH 7.5 and 10 mM at pH 8.0, respectively.